There is no organ in the body as dependent on a continuous supply of blood as the brain. If cerebral blood flow (CBF) is interrupted, brain function ceases within seconds and irreversible damage to its cellular constituents ensues within minutes. In elderly patients, increasing evidence suggests that vascular risk factors and lowered CBF have a major role in the development of Alzheimer's disease (AD) and possibly even trigger the onset of dementia. We have discovered an over-abundance of two interactive transcription factors in vascular smooth muscle cells (VSMC) from pial cerebral arteries of patients with AD which critically regulate CBF. The factors, serum response factor (SRF) and myocardin (MYOCD), are potent inducers of a contractile gene program linked to VSMC function. Consequently, AD-VSMC display elevated expression of several contractile proteins that effect a hypercontractile state in these cells. Based on these novel findings, we hypothesize that elevated SRF/MYOCD activity in VSMC of small cerebral arteries leads to a hypercontractile phenotype, which contributes to CBF reductions and neurovascular uncoupling in AD. In this Phase I application, we endeavor to further validate these findings and optimize a high-throughput screening assay for SRF/MYOCD-dependent gene transcription. To this end, we have delineated three milestones we are confident we can achieve as a critical foundation for a Phase II application that we have simultaneously submitted in this Fast Track proposal. Milestone 1 will assess the ability of MYOCD to elicit an AD-VSMC hypercontractile phenotype in age-matched control VSMC. Milestone 2 will ascertain whether the AD-VSMC hypercontractile phenotype can be normalized with an RNAi to SRF. In Milestone 3, we will optimize a recently developed large-scale luciferase assay for human cells in preparation for studies described in Phase II. The finding that two interactive transcription factors may underlie the known brain hypoperfusion and cognitive decline in AD represents a new paradigm for therapeutic interventions. The milestones to be addressed here will provide a vital platform for aims delineated in Phase II of this Fast Track Application in which a diverse small molecule library of compounds is to be screened for organic chemicals that specifically disrupt the SRF/MYOCD interaction thereby normalizing VSMC hypercontraction and, by extension, brain hypoperfusion and dysregulated CBF in AD. [unreadable] [unreadable] [unreadable]